WO2021076592A1 - Protéines de liaison à l'adn permettant de déplacer des facteurs de transcription endogènes liés à des régions régulatrices de gènes - Google Patents

Protéines de liaison à l'adn permettant de déplacer des facteurs de transcription endogènes liés à des régions régulatrices de gènes Download PDF

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WO2021076592A1
WO2021076592A1 PCT/US2020/055534 US2020055534W WO2021076592A1 WO 2021076592 A1 WO2021076592 A1 WO 2021076592A1 US 2020055534 W US2020055534 W US 2020055534W WO 2021076592 A1 WO2021076592 A1 WO 2021076592A1
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cell
dbp
sequence
nucleic acid
gene
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PCT/US2020/055534
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English (en)
Inventor
Jennifer M. CHERONE
Alister PW FUNNELL
John A. Stamatoyannopoulos
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Altius Institute For Biomedical Sciences
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Priority to US17/764,497 priority Critical patent/US20230227513A1/en
Priority to EP20877985.0A priority patent/EP4045649A4/fr
Publication of WO2021076592A1 publication Critical patent/WO2021076592A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • Modulation of gene expression is useful in studying protein function as well as in treating diseases.
  • Anemia a red blood cell disorder
  • Anemia can be defined as a reduction in the ability of blood to transport oxygen.
  • the majority of red blood cell disorders are caused by genetic defects that result in abnormal hemoglobin, such as, sickle cell syndromes; low hemoglobin, such as, thalassemia syndromes; or both, e.g., syndromes associated with unstable hemoglobins.
  • Fetal globin also known as hemoglobin gamma “HBG” or gamma globin
  • HbF fetal hemoglobin
  • HBB hemoglobin beta
  • Fetal globin performs the same function as beta globin, and can combine with the alpha chains to generate a healthy form of hemoglobin.
  • beta thalassemias are syndromes resulting from mutations, which produce a deficiency of beta globin chains.
  • beta thalassemia the unmatched alpha globin chains aggregate inside red blood cells (RBCs) and their progenitors, causing the premature destruction of RBCs and RBC progenitors, which results in anemia, transfusion- dependence, iron overload, organ failure, and early death.
  • RBCs red blood cells
  • SCD sickle cell disease
  • HbS sickling hemoglobin
  • the sickled RBCs undergo hemolysis, while adhesive sickled RBCs occlude the microcirculation, provoking widespread tissue ischemia and organ infarction.
  • the natural history of SCD is marked by painful crises, acute chest syndrome, and eventual potentially life-threatening sequelae, including renal insufficiency, retinitis, osteonecrosis, osteomyelitis, aplastic crises, functional asplenism, stroke, priapism, and severe pulmonary hypertension.
  • the disclosure herein provides novel methods and compositions for modulating expression of target genes such as increasing expression of fetal hemoglobin in a patient with a blood disorder, including beta thalassemias and sickle cell disease.
  • the present disclosure provides methods and compositions for modulating expression of a target gene in a cell by reducing binding of an endogenous transcription factor to a regulatory sequence of the target gene.
  • the method includes introducing into the cell a DNA binding polypeptide (DBP) that binds a sequence in regulatory region of a target gene bound by a transcription factor (TF), thereby displacing the TF and modulating expression of the target gene.
  • DBP DNA binding polypeptide
  • the DBP may be designed to bind a sequence comprising the binding site for the TF and additional nucleotides present on one or both sides of the sequence. Accordingly, the DBP specifically binds to binding site for the TF in the target gene but not in other genes that are also regulated by binding of the TF but do not include the nucleotides present on one or both sides of the sequence.
  • the binding site for the TF is a sequence that has previously been identified as being associated with activity of the TF based on reduced activity of the TF when the sequence includes a single nucleotide polymorphism (SNP) or a mutation that may reduce binding of the TF.
  • SNP single nucleotide polymorphism
  • the DBP includes a plurality of RUs that are arranged from N- terminus to C-terminus to bind to a sequence bound by a TF and additional nucleotides present on one or both sides of the sequence.
  • a recombinant DBP that includes a plurality of RUs that are arranged from N-terminus to C-terminus to bind to a sequence bound by the TF, ZBTB7A in the fetal g-globin gene promoter.
  • the RUs may be derived from a TALE protein and the DBP may include at least 9.5 RUs, which include 9 RUs and a terminal half-repeat unit (0.5 RU).
  • the DBP displaces ZBTB7A from the fetal g-globin gene promoter and relieves suppression of the fetal g-globin gene.
  • the DBP also decreases expression of adult hemoglobin B (HBB).
  • HBB adult hemoglobin B
  • a cell line comprising a stable expression of the recombinant
  • the cell line may be produced by stable integration into the genome of the cells of a nucleic acid encoding the DBP.
  • the cell line may be administered to a subject in need thereof.
  • FIGS. 1A-1D Transiently expressed TALEs with or without Fokl activate HBG expression in a position-dependent manner.
  • FIG. 1A Relative expression of HBG compared to HBB in HUDEP-2 cells at 12, 24, or 48 hours post-transfection of Bl, D2, B1+D2 TALE with the Fokl domain mRNA, or no mRNA (mock).
  • FIG. IB Relative expression of HBG compared to HBB in HUDEP-2 cells at 48 hours post-transfection of Bl with the Fokl domain (Bl), Bl with no effector domain (B1NF), GFP, or no mRNA.
  • FIG. ID Relative expression of HBG compared to HBB in HUDEP-2 cells at 48 hours post-transfection of Bl with the Fokl domain (Bl), Bl with no effector domain (B1NF), GFP, or no mRNA.
  • FIG. 1C Immunofluorescent imaging of FLAG (FLAG IF) at 6, 24, or 48 hours post-transfection of no mRNA, AAVS1R TALEN, Bl, B1NF, B7, B7NF, Dll, D11NF (NF means no effector domain). Significance was assessed by t-test. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • FIGS. 2A-2D Stable expression of the A11 TALE results in globin switching at the RNA and protein level.
  • FIG. 2B Expression of HBG out of total globin (HBG + HBB) in clonal cell lines generated in three independent transfections, sorts, and expansion experiments.
  • FIG. 2C Expression of HBG out of total globin (HBG + HBB) in clonal cell lines generated in three independent transfections, sorts, and expansion experiments.
  • FIG. 2D FLAG IF of 5 most highly expressing All clones, WT or Bl clone and corresponding HBG out of total globin expression measured by TaqMan qPCR.
  • FIG. 3 Cassette for expression of TALE designed for integration into the
  • FIG. 4 Increased expression of HBG is accompanied by a decreased expression of HBB.
  • the present disclosure provides methods and compositions for modulating expression of a target gene in a cell by reducing binding of an endogenous transcription factor to a regulatory sequence of the target gene.
  • the method includes introducing into the cell a DNA binding polypeptide (DBP) that binds a sequence in regulatory region of a target gene bound by a transcription factor (TF), thereby displacing the TF and modulating expression of the target gene.
  • DBP DNA binding polypeptide
  • the DBP may be designed to bind a sequence comprising the binding site for the TF and additional nucleotides present on one or both sides of the sequence. Accordingly, the DBP specifically binds to binding site for the TF in the target gene but not in other genes that are also regulated by binding of the TF but do not include the nucleotides present on one or both sides of the sequence.
  • the binding site for the TF is a sequence that has previously been identified as being associated with activity of the TF based on reduced activity of the TF when the sequence includes a single nucleotide polymorphism (SNP) or a mutation that may reduce binding of the TF.
  • SNP single nucleotide polymorphism
  • the DBP includes a plurality of RUs that are arranged from N- terminus to C-terminus to bind to a sequence bound by a TF and additional nucleotides present on one or both sides of the sequence.
  • a recombinant DBP that includes a plurality of RUs that are arranged from N-terminus to C-terminus to bind to a sequence bound by the TF, ZBTB7A in the fetal g-globin gene promoter.
  • the RUs may be derived from a TALE protein and the DBP may include at least 9.5 RUs, which include 9 RUs and a terminal half-repeat unit (0.5 RU).
  • the DBP displaces ZBTB7A from the fetal g-globin gene promoter and relieves suppression of the fetal g-globin gene.
  • the DBP also decreases expression of adult hemoglobin B (HBB).
  • HBB adult hemoglobin B
  • a cell line comprising a stable expression of the recombinant
  • the cell line may be produced by stable integration into the genome of the cells of a nucleic acid encoding the DBP.
  • the cell line may be administered to a subject in need thereof.
  • polypeptide derived in the context of a polypeptide refers to a polypeptide that has a sequence that is based on that of a protein from a particular source (e.g., an animal pathogen such as Legionella or a plant pathogen such as Xanthomonas).
  • a polypeptide derived from a protein from a particular source may be a variant of the protein from the particular source (e.g., an animal pathogen such as Legionella or a plant pathogen such as Xanthomonas).
  • a polypeptide derived from a protein from a particular source may have a sequence that is modified with respect to the protein’s sequence from which it is derived.
  • a polypeptide derived from a protein from a particular source shares at least 30% sequence identity with, at least 40% sequence identity with, at least 50% sequence identity with, at least 60% sequence identity with, at least 70% sequence identity with, at least 80% sequence identity with, or at least 90% sequence identity with the protein from which it is derived.
  • the DBP disclosed herein may be derived from a nucleic acid binding domain of a DNA binding protein of an animal or plant pathogen.
  • the term “modular” as used herein in the context of a nucleic acid binding domain e.g., a modular animal pathogen derived DNA binding polypeptide (MAP-DBP) indicates that the plurality of repeat units present in the DBP can be rearranged and/or replaced with other repeat units and can be arranged in an order such that the DBP binds to the target nucleic acid. For example, any repeat unit in a modular nucleic acid binding domain can be switched with a different repeat unit.
  • modularity of the nucleic acid binding domains disclosed herein allows for switching the target nucleic acid base for a particular repeat unit by simply switching it out for another repeat unit. In some aspects, modularity of the nucleic acid binding domains disclosed herein allows for swapping out a particular repeat unit for another repeat unit to increase the affinity of the repeat unit for a particular target nucleic acid. Overall, the modular nature of the nucleic acid binding domains disclosed herein enables the development of DBP that can precisely target any nucleic acid sequence of interest.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones.
  • the terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminus methionine residues; immunologically tagged proteins; and the like.
  • the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids.
  • the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids fused to a heterologous amino acid sequence.
  • heterologous refers to two components that are defined by structures derived from different sources.
  • a “heterologous” polypeptide may include operably linked amino acid sequences that are derived from different polypeptides (e.g., a NBD and a functional domain derived from different sources).
  • a “heterologous” polynucleotide may include operably linked nucleic acid sequences that can be derived from different genes.
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence or both).
  • a regulatory element e.g., a promoter
  • heterologous can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.
  • operably linked refers to linkage between molecules to provide a desired function.
  • “operably linked” in the context of nucleic acids refers to a functional linkage between nucleic acid sequences.
  • a nucleic acid expression control sequence such as a promoter, signal sequence, or array of transcription factor binding sites
  • the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • “operably linked” refers to a functional linkage between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide.
  • cleavage refers to the breakage of the covalent backbone of a nucleic acid, e.g., a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single- stranded cleavage and double- stranded cleavage are possible, and double- stranded cleavage can occur as a result of two distinct single- stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain aspects, the polypeptides provided herein are used for targeted double-stranded DNA cleavage.
  • a “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
  • a “target nucleic acid,” “target sequence,” or “target site” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule, such as, the DBP disclosed herein will bind.
  • the target nucleic acid may be present in inside a cell.
  • a target nucleic acid may be present in a regulatory region, e.g., promoter sequence, of a target gene whose expression is to be modulated by the DBP.
  • exogenous molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods.
  • An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule, e.g. a gene or a gene segment lacking a mutation present in the endogenous gene.
  • An exogenous nucleic acid can be present in an infecting viral genome, a plasmid or episome introduced into a cell.
  • lipid-mediated transfer i.e., liposomes, including neutral and cationic lipids
  • electroporation direct injection
  • cell fusion cell fusion
  • particle bombardment particle bombardment
  • calcium phosphate co-precipitation DEAE-dextran-mediated transfer
  • viral vector-mediated transfer viral vector-mediated transfer.
  • an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, shRNA, RNAi, miRNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.
  • Modulation of gene expression refers to a change in the activity of a gene.
  • Modulation of expression can include, but is not limited to, gene activation and gene repression.
  • Genome editing e.g., cleavage, alteration, inactivation, donor integration, random mutation
  • Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a polypeptide or has not been modified by a polypeptide as described herein. Thus, gene inactivation may be partial or complete.
  • patient or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal).
  • a disease, disorder or condition, or a symptom thereof initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • prevent refers to a course of action (such as administering a polypeptide or a nucleic acid encoding the polypeptide or a cell comprising the nucleic acid encoding the polypeptide or expressing the polypeptide) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a subject predisposed to having a particular disease, disorder or condition.
  • the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
  • therapeutically effective amount refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects.
  • conjugating refers to an association of two entities, for example, of two molecules such as two proteins, two domains (e.g., a binding domain and a cleavage domain), or a protein and an agent, e.g., a protein binding domain and a small molecule.
  • the association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage or via non-covalent interactions. In some aspects, the association is covalent. In some aspects, two molecules are conjugated via a linker connecting both molecules.
  • the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein.
  • a polypeptide linker e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein.
  • conjugated proteins may be expressed as a fusion protein.
  • sequence refers to a sequence representing the most frequent nucleotide/amino acid residues found at each position in a plurality of similar sequences.
  • a consensus sequence is determined by sequence alignment in which similar sequences are compared to each other.
  • a consensus sequence of a protein can provide guidance as to which residues can be substituted without significantly affecting the function of the protein.
  • genome modifying proteins refer to nucleic acid binding domains and functional domains which cooperate to modify genome or epigenome is a cell.
  • genome modifying proteins include but are not limited to nucleic acid binding proteins comprising modular repeat units, nucleic acid binding proteins comprising zinc fingers, functional domains such as labels, tags, polypeptides having nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, e.g., nucleases, transcriptional activators, transcriptional repressors, chromatin modifying protein, and the like.
  • Genome modifying proteins also encompass a single polypeptide comprising a nucleic acid binding domain and functional domain or two or more polypeptides, where a first polypeptide comprises a nucleic acid binding domain and a second polypeptide comprises a functional domain and wherein the first and second polypeptide associate with each other via a non-covalent interaction, such as, via a interactions mediated by first and second members of a heterodimer, where one of the first and second polypeptide is conjugated to the first member and the other polypeptide is conjugated to the second member.
  • a “fusion protein” includes a first protein moiety, e.g., a nucleic acid binding domain, having a peptide linkage with a second protein moiety.
  • the fusion protein is encoded by a single fusion gene.
  • Domain is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.
  • gene therapy refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide.
  • introduction of genetic material into the cell’s genome for the purpose of expressing a therapeutic polypeptide is considered gene therapy.
  • the present disclosure provides a method for modulating expression of a target gene in a cell, the method comprising introducing into the cell a DNA binding polypeptide (DBP) that binds a sequence in regulatory region of a gene bound by a transcription factor (TF), thereby displacing the TF and modulating expression of the gene.
  • DBP DNA binding polypeptide
  • the sequence include a binding site for a TF that has also previously been identified as being associated with activity of the TF based on reduced activity of the TF when the binding site includes or is adjacent to a single nucleotide polymorphism (SNP) or a mutation.
  • the binding site may include or is adjacent to SNPs or mutations that lead to an increased expression of the protein encoded by the gene, which may be indicative of reduced binding of a TF, such as a transcriptional repressor, to the binding site.
  • the binding site may include or is adjacent to SNPs or mutations that lead to a decreased expression of the protein encoded by the gene, which may be indicative of reduced binding of a TF, such as a transcriptional activator, to the binding site.
  • it may be desirable to displace the TF from the binding site in the regulatory region of a target gene.
  • the method for modulating expression of the target gene in a cell includes introducing into the cell a DBP that binds to the binding site and additional nucleotides present adjacent the binding site, e.g., nucleotides flanking the binding site.
  • binding site refers to a core sequence that is required for binding of a TF to the regulatory region of a gene and modulate gene expression.
  • a binding site is usually less than 10 nucleotides in length and more commonly 4-8 nucleotides in length.
  • a TF may bind to more than one binding site in the regulatory region of a gene.
  • the DBP may bind to a binding site as well as nucleotides adjacent the binding site to increase the binding specificity of the DBP such that it binds to the binding site for the TF for a target gene but not to other genes that also including a binding site for the TF but include different nucleotide sequences adjacent the binding site.
  • the DBP may bind to at least a 12 nucleotides long sequence comprising the sequence bound by the TF. In certain aspects, the DBP may bind to at least a 14, 16, 18, 20, 24, 26, 28, 30, or up to 45 nucleotides long sequence comprising the sequence bound by the TF. In certain aspects, the DBP may bind to the a binding site for a transcriptional activator and the introducing results in reduced expression of the gene. In certain aspects, the DBP may bind to the a binding site for a transcriptional repressor and the introducing results in increased expression of the gene.
  • the DBP is introduced into the cell as a nucleic acid encoding the DBP.
  • the nucleic acid may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
  • the sequence of the nucleic acid is codon optimized for expression in a human cell. Methods and compositions for introducing a nucleic acid into a cell are described in detail in subsequent sections of this disclosure.
  • the cell is a human cell, such as, a cancer cell, an ex vivo cell obtained from a subject, a stem cell, a hematopoietic stem cell, or the like.
  • a target gene may fetal hemoglobin gamma.
  • introducing the DBP into a cell may include administering the polypeptide or a nucleic acid encoding the polypeptide to a subject in need thereof.
  • the DBPs of the present disclosure include modular units that mediate binding to a nucleotide sequence in the regulatory region of a target gene in a cell.
  • the modular units may be derived from DNA binding domains of proteins known to specifically bind to a nucleotide sequence.
  • Such modular units include zing fingers, megaTAL, repeat units from TALE protein, repeat units from DNA binding proteins from animal pathogens, and the like.
  • the DBP may bind to the binding sequence of the TF
  • the DBP may bind to sequences adjacent the binding site.
  • the DBP may bind to the sequence CCTCTTGGGGGCCCC (SEQ ID NO: 1) in the promoter region of the fetal g-globin gene and displace ZBTB7A and increase expression of the fetal g-globin gene.
  • the DBP may bind to the sequence ATCCTCTTGGGGGCCCC (SEQ ID NO: 2) in the promoter region of the fetal g-globin gene and displace ZBTB7A and increase expression of the fetal g-globin gene.
  • the DBP may bind to the sequence CCTCTTGGGGGCCCCTTCCC (SEQ ID NO: 3) in the promoter region of the fetal g-globin gene and displace ZBTB7A and increase expression of the fetal g-globin gene.
  • the DBP includes at least ten repeat units (RUs) ordered from N-terminus to C- terminus of the DBP to specifically bind to sequence bound by a TF as disclosed herein, wherein each of the RUs comprises the sequence:
  • Xi-11 is a chain of 11 contiguous amino acids
  • X14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids
  • X12X13 is selected from:
  • RA for recognition of A or T or G or C
  • (* ) means that the amino acid at X13 is absent
  • the DBP displaces the TF from the regulatory region (e.g. promoter or enhancer region) of a target gene and modulates expression of the target gene.
  • the DBP includes a half-repeat unit (0.5RU) as the last RU such that the DBP includes at least 10.5 RUs.
  • a half-repeat unit may have the amino acid sequence X 1X12X13X14-19, 20, or 21 (SEQ ID NO: 5), wherein: [0073] Xi-ii is a chain of 11 contiguous amino acids,
  • X14-20 or 21 or 22 is a chain of 7, 8 or 9 contiguous amino acids
  • X 12 X 13 is selected from:
  • Xi-ii is at least 80% identical, at least 90% identical, or 100% identical to LTPEQVVAIAS (SEQ ID NO: 6).
  • Xi 4-20 or 2i or 22 is at least 80% identical to GGRPALE (SEQ ID NO: 7).
  • the present disclosure provides a DBP that includes a plurality of repeat units
  • RUs comprising a 33-36 amino acid long sequence having at least 80% sequence identity to the amino acid sequence:
  • LTPDQVVAIASX 12 X 13 GGKQALETVQRLLPVL QDHG (SEQ ID NO: 8), or having the sequence of SEQ ID NO:l with one or more conservative amino acid substitutions thereto;
  • X12X13 is HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN, NI, KI, RI, HI, SI, NG, HG, KG, RG, RD, SD, HD, ND, KD, YG, YK, NV, HN, H*, HA, KA, N*, NA, NC, NS, RA, Cl, or S*, where (*) means X13 is absent
  • the RU may comprise a 33-36 amino acid long sequence having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or more identical to SEQ ID NO: 8.
  • the RUs and the half-RU, if present, are derived from
  • Xi-11 is at least 80%, at least 90%, or 100% identical to LTPEQVVAIAS (SEQ ID NO: 6), LTPAQVVAIAS (SEQ ID NO: 9), LTPDQ V V AIAN (SEQ ID NO: 10), LTPDQVVAIAS (SEQ ID NO: 11), LTPYQVVAIAS (SEQ ID NO: 12), LTREQVVAIAS (SEQ ID NO: 13), or LSTAQVVAIAS (SEQ ID NO: 14).
  • Xi4-2o or 21 or 22 is at least 80%, at least 90%, at least 95%, or 100% identical to GGKQ ALET V QRLLP VLCQDHG (SEQ ID NO: 15), GGKQ AL AT V QRLLPVLCQDHG (SEQ ID NO: 16), GGKQALETVQRVLPVLCQDHG (SEQ ID NO: 17), or GGKQ ALET VQRVLPVLCQDHG (SEQ ID NO: 17).
  • the DBP may include a plurality of RUs ordered from N- terminus to C-terminus of the DBP to bind a nucleic acid sequence comprising the binding site for a TF in the regulatory region of a target gene.
  • the DBP may include 10, 11, 12,
  • the DBP may include a plurality of RUs of naturally occurring transcription activator like effector (TALE) proteins, such as RUs from Xanthomonas or Ralstonia TALE proteins.
  • TALE transcription activator like effector
  • one or more RUs in a DBP may be at least 80%, 85%, 90%,
  • Percent identity between a pair of sequences may be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. Only internal gaps are included in the length, not gaps at the sequence ends.
  • conservative amino acid substitution refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A,
  • Conservative amino acid substitutions may preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of proteins from different species or from a consensus sequence based on a plurality of proteins having the same or similar function.
  • the disclosed DBP may include a nuclear localization sequence
  • the disclosed DBP may include a half-RU or a partial RU that is 15-20 amino acid long sequence. Such a half-RU may be included after the last RU present in the DBP and may be derived from a RU identified in Xanthomonas or Ralstonia TALE protein.
  • the disclosed DBP may include an N-terminal domain. The N-terminal domain may be the N-cap domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the disclosed DBP may include a C-terminal domain.
  • the C-terminal domain may be a C-cap domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the N-terminal domain may be at least 80%, 85%, 90%, 91%,
  • This amino acid sequence includes a M added to the N-terminus which is not present in the wild type N-cap region of a Xanthomonas TALE protein.
  • This amino acid sequence is generated by deleting amino acids N+288 through N+137 of the N-terminus region of a TALE protein, adding a M, such that amino acids N+136 through N+l of the N- terminus region of the TALE protein are present.
  • the N-terminus can be truncated such that the fragment of the N- terminus includes amino acids from position 1 (N) through position 120 (K) of the naturally occurring Xanthomonas spp.- derived protein as follows:
  • the N-cap region can be truncated such that the fragment of the
  • N-terminus includes amino acids from position 1 (N) through position 115 (S) of the naturally occurring Xanthomonas spp.- derived protein as follows:
  • the N-cap region can be truncated and may include amino acids from position 1 (N) through position 110 (H) of the naturally occurring Xanthomonas spp.- derived protein as follows:
  • the DBP may include a C-cap region at C-terminus of the recombinant polypeptide which C-cap region is derived from the C-cap region of a Xanthomonas TALE protein.
  • the C-terminal domain may be at least 80%, 85%, 90%, 91%,
  • the RUs are derived from Xanthomonas TALEs.
  • T thymine
  • the natural TALE-binding sites begin with a thymine (T), which may be specified by a cryptic signal within the non-repetitive N-terminus region of the TALE polypeptide; in some cases this region may be referred to as repeat 0.
  • TALE binding sites do not necessarily have to begin with a thymine (T) and recombinant DBP disclosed herein may target DNA sequences that begin with T, A, G or C.
  • the recombinant DBP disclosed herein may target DNA sequences that begin with T.
  • the tandem repeat of TALE RUs ends with a half-length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full length TALE RU and this half repeat may be referred to as a half-monomer, a half RU, or a half repeat. Therefore, it follows that the length of the DNA sequence being targeted by DBP derived from TALEs is equal to the number of full RUs plus two.
  • DBP may be engineered to include X number (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) full length RUs that are specifically ordered or arranged to target nucleic acid sequences of X+2 length (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides, respectively), with the N-terminus region binding “T” and the last RU being a half-repeat.
  • X number e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26
  • a Xanthomonas spp. -derived repeat units can have a sequence of LTPDQVVAIASNHGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 21) comprising an RVD of NH, which recognizes guanine.
  • a Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 22) comprising an RVD of NG, which recognizes thymidine.
  • a Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 23) comprising an RVD of NI, which recognizes adenosine.
  • a Xanthomonas spp.-derived repeat units can have a sequence of LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 24) comprising an RVD of HD, which recognizes cytosine.
  • the DBP comprises RUs that binds to the nucleotide sequence: CCTCTTGGGGGCCCC (SEQ ID NO: 1) in the regulatory region of the fetal D-globin gene and induces expression of HBG.
  • X12X13 in the RUs from N-terminus to C- terminus are HD, HD, NG, HD, NG, NG, NH, NH, NH, NH, HD, HD, HD, and HD, wherein the last RU is a half-RU.
  • the DBP comprises RUs that binds to the nucleotide sequence: ATCCTCTTGGGGGCCCC (SEQ ID NO: 2) in the regulatory region of the fetal D-globin gene and induces expression of HBG.
  • X12X13 in the RUs from N-terminus to C- terminus are NI, NG, HD, HD, NG, HD, NG, NG, NH, NH, NH, HD, HD, HD, and HD, wherein the last RU is a half-RU.
  • the DBP comprises RUs that binds to the nucleotide sequence: CCTCTTGGGGGCCCCTTCCC (SEQ ID NO: 3) in the regulatory region of the fetal D-globin gene and induces expression of HBG.
  • X12X13 in the RUs from N-terminus to C-terminus are HD, HD, NG, HD, NG, NG, NH, NH, NH, NH, NH, HD, HD, HD, HD, NG, NH, NH, NH, HD, HD, HD, NG, NH,
  • NG NG, HD, HD, and HD, wherein the last RU is a half-RU.
  • expression of the target gene can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% by using a DNA binding polypeptide that displaces a transcriptional activator as compared to untreated cells.
  • expression of the target gene can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% by using a DNA binding polypeptide that displaces a transcriptional repressor as compared to untreated cells.
  • modulation of gene expression e.g., repression or activation of the target gene
  • a DNA binding polypeptide that displaces a transcriptional factor can last for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, 1 days to 3 days, 3 days to 5 days, 5 days to 7 days, 7 days to 9 days, 9 days to 11 days, 11 days to 13 days, 13 days to 15 days, 15 days to 17 days, 17 days to 19 days, 19 days to 21 days, 21 days to 23 days
  • the RUs and one or both N-Cap and C-Cap regions may be derived from a transcription activator like effector-like protein (TALE-like protein) of Ralstonia solanacearum.
  • TALE-like protein transcription activator like effector-like protein
  • Repeat units derived from Ralstonia solanacearum can be 33-35 amino acid residues in length.
  • the repeat can be derived from the naturally occurring Ralstonia solanacearum TALE-like protein.
  • the RUs may have the sequence X1-11X12X13X14-33 , 34 , or 35 (SEQ ID NO: 25) , where Xi-11 is a chain of 11 contiguous amino acids, X14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X12X13 is RVD and is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*
  • Xl-11 may include a stretch of amino acids at least 80%, at least 90%, or a 100% identical to the Xl-11 residues of the following RUs from Ralstonia.
  • X14-33 or 34 or 35 may include a stretch of 20, 21 , or 22 amino acids at least 80%, at least 90%, or a 100% identical to the X14-33 or 34 or 35 residues of the following RUs from Ralstonia: LDTEQ V V AI ASHN GGKQ ALEA VK ADLLDLLG APY V (SEQ ID NO: 26), LNTEQ V V A VAS NKGGKQ ALE A V G AQLL ALRA VP YE (SEQ ID NO:
  • a Ralstonia solanacearum- repeat unit can have at least 80% sequence identity with any one of the Ralstonia RUs provided herein.
  • the DBP may include a N-cap region at the N-terminus which may be present immediately adjacent the first RU or may be linked to the first RU via a linker.
  • an DBP of the present disclosure can have the full length naturally occurring N- terminus of a naturally occurring Ralstonia solanacearum-demed protein.
  • any truncation of the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-demed protein can be used at the N-terminus of a DBP of the present disclosure.
  • amino acid residues at positions 1 (H) to position 137 (F) of the naturally occurring Ralstonia solanacearum-demed protein N-terminus can be used as the N- cap region.
  • the truncated N-terminus from position 1 (H) to position 137 (F) can have a sequence as follows:
  • the naturally occurring N- terminus of Ralstonia solanacearum can be truncated to any length and used as the N-cap of the engineered DNA binding polypeptide.
  • the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to include amino acid residues at position 1 (H) to position 120 (K) as follows:
  • Ralstonia solanacearum can be truncated amino acid residues to include positions 1 to 115 and used at the N-cap of the engineered DNA binding domain.
  • the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 50, 1 to 70,
  • N-cap region derived from Xanthomonas TALE the amino acid residues are numbered backward from the first repeat unit such that the amino acid (H in this case) of the N-cap adjacent the first RU is numbered 1 while the N-terminal amino acid of the N-cap is numbered 137 (and is F in this case) or 120 (and is K in this case).
  • the N-cap referred to as the amino terminus or the “NH2” domain, can recognize a guanine.
  • the N-cap can be engineered to bind a cytosine, adenosine, thymidine, guanine, or uracil.
  • an DBP of the present disclosure can include a plurality of RUs followed by a final single half-repeat also derived from Ralstonia solanacearum.
  • the half repeat can have 15 to 23 amino acid residues, for example, the half repeat can have 19 amino acid residues.
  • the half-repeat can have a sequence as follows:
  • an DBP of the present disclosure can have the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derl ved protein as a C-cap region that is conjugated to the last RU.
  • any truncation of the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-demed protein can be used as the C-cap.
  • the DBP can comprise amino acid residues at position 1 (A) to position 63 (S) as follows:
  • AIEAHMPTLRQASHSLSPERVAAIACIGGRSAVEAVRQGLPVKAIRRIRREKAPVAGPPP AS (SEQ ID NO: 34) of the naturally occurring Ralstonia solanacearum-demed protein C- terminus.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to any length and used as the C-cap of the DBP.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63 and used as the C-terminus of the DBP.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated amino acid residues at positions 1 to 50 and used as the C-cap of the DBP.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40,
  • Exemplary sequences of domains of a DBP as disclosed herein are as follows:
  • the present disclosure provides DNA binding polypeptide in which the repeat units can be derived from a Legionellales bacterium, a species of the genus of Legionella, such as L. quateirensis or L. maceachernii, the genus of Burkholderia, the genus of Paraburkholderia, or the genus of Francisella.
  • a Legionellales bacterium a species of the genus of Legionella, such as L. quateirensis or L. maceachernii, the genus of Burkholderia, the genus of Paraburkholderia, or the genus of Francisella.
  • the RUs may have the sequence X1-11X12X13X14-33, 34, or 35 (SEQ ID NO: 36), where Xi-11 is a chain of 11 contiguous amino acids, X14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X12X13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, HN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, HA, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA,
  • Xl-11 may include a stretch of amino acids at least 80%, at least 90%, or a 100% identical to the Xl-11 residues of the following RUs from animal pathogens, Legionella, Burkholderia, Paraburkholderia, or Francisella.
  • X14-33 , 34 , or 35 may include a stretch of 20, 21, or 22 amino acids at least 80%, at least 90%, or a 100% identical to the X14-33 , 34 , or 35 residues of the RUs from animal pathogens, Legionella (e.g., L. quateirensis or L. maceachernii ), Burkholderia, Paraburkholderia, or Francisella listed below.
  • Residues X12X13 of the RU may a base contacting residues (BCR) as listed in the table 8 and may be chosen based upon the target nucleic acid sequence.
  • the last RU in the DBP may be a half RU.
  • the half RU may include a sequence that is at least 80%, at least 90%, at least 95% or a 100% identical to the half RU from L. quateirensis (FNAEQIVRMV S X12X13 GGSKNL; SEQ ID NO: 138).
  • the half RU may include a sequence that is at least 80%, at least 90%, at least 95% or a 100% identical to the half RU from Francisella (YNKKQIVLIAS X12X13 SGG; SEQ ID NO: 139)
  • the polypeptide comprises an N-cap region, where the C- terminus (i.e., the last amino acid) of the N-cap region is covalently linked to the N-terminus (i.e., the first amino acid) of the first RU of the DBP either directly or via a linker.
  • the N-cap region is the N-terminus of L. quateirensis protein and may have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, 95%, or 99%, or a 100%) identical to the amino acid sequence:
  • the N-cap region is a N-terminal domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the polypeptide comprises a C-cap region, where the N- terminus (i.e., the first amino acid) of the C-terminal domain is covalently linked to the C- terminus (i.e., the last amino acid) of the last RU or the half-repeat unit, if present, in the DBP either directly or via a linker.
  • the C-cap region is the C-terminal domain of L. quateirensis protein and may have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, 95%, or 99%, or a 100%) identical to the amino acid sequence:
  • the C-cap region has the amino acid sequence ALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETP PKP (SEQ ID NO: 142).
  • the C-cap region domain is a C-terminal domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the present disclosure provides DNA binding domains in which the repeat units, the N-cap, and the C-ap can be derived from any one of Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella.
  • the present disclosure provides a DNA binding domain wherein the plurality of repeat units are selected from any one of the RUs as provided herein and can further comprise an N-cap and/or C-cap as provided herein.
  • a DBP as disclosed herein can be associated with a functional domain as described in the preceding sections.
  • the functional domain can provide different types of activity, such as genome editing, gene regulation (e.g., activation or repression), or visualization of a genomic locus via imaging.
  • the functional domain is heterologous to the DBP. Heterologous in the context of a functional domain and a DBP as used herein indicates that these domains are derived from different sources and do not exist together in nature.
  • the nuclease can be a cleavage half domain, which dimerizes to form an active full domain capable of cleaving DNA.
  • the nuclease can be a cleavage domain, which is capable of cleaving DNA without needing to dimerize.
  • a nuclease comprising a cleavage half domain can be an endonuclease, such as Fokl or Bfil.
  • two cleavage half domains e.g., Fokl or Bfil
  • Fokl or Bfil two cleavage half domains
  • a nuclease domain fused to a DBP can be an endonuclease or an exonuclease.
  • An endonuclease can include restriction endonucleases and homing endonucleases.
  • An endonuclease can also include SI Nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, or yeast HO endonuclease.
  • An exonuclease can include a 3’ -5’ exonuclease or a 5 ’-3’ exonuclease.
  • exonuclease can also include a DNA exonuclease or an RNA exonuclease.
  • exonuclease includes exonucleases I, II, III, IV, V, and VIII; DNA polymerase I, RNA exonuclease 2, and the like.
  • a nuclease domain fused to a DBP as disclosed herein can be a restriction endonuclease (or restriction enzyme).
  • a restriction enzyme cleaves DNA at a site removed from the recognition site and has a separate binding and cleavage domains.
  • such a restriction enzyme is a Type IIS restriction enzyme.
  • DBP as disclosed herein can be linked to a gene regulating domain.
  • a gene regulation domain can be an activator or a repressor.
  • a DBP as disclosed herein can be linked to an activation domain, such as VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldbl self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta).
  • a DBP can be linked to a repressor, such as KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
  • a repressor such as KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
  • a repressor such as KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DN
  • a DBP can be linked to a chromatin-modifying protein, such as lysine-specific histone demethylase 1 (LSD1).
  • LSD1 lysine-specific histone demethylase 1
  • a DBP can be linked to a protein that is capable of recruiting other proteins, such as KRAB.
  • the DNA modifying protein e.g., DNMT3a
  • proteins capable of recruiting other proteins e.g., KRAB
  • DBP linked to a DNA modifying protein e.g., DNMT3a
  • a domain capable of recruiting other proteins e.g., KRAB, a domain found in transcriptional repressors, such as Koxl
  • DBP-GR DBP-gene regulator
  • DBP-TF DBP -transcription factor
  • the functional domain may be an imaging domain, e.g., a fluorescent protein, biotinylation reagent, tag (e.g., 6X-His or HA).
  • a DBP can be linked to a fluorophore, such as Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7, GFP, or mCHERRY.
  • the DBP is not fused with a functional domain having a genome modifying activity, such as, cleavage activity, DNA methylation activity, chromatin modifying protein, transcriptional activation, or transcriptional repression.
  • a genome modifying activity such as, cleavage activity, DNA methylation activity, chromatin modifying protein, transcriptional activation, or transcriptional repression.
  • a cell that expresses the DBP disclosed herein may be a mammalian cell such as a stem cell (e.g., human embryonic stem cell or induced pluripotent stem cell), human hematopoietic stem cell “HSC” (e.g. CD34 + HSC), hematopoietic progenitor cell (HPC), a cell in the erythroid lineage, a lymphocyte, a T-cell, CAR-T cells, a cancer cell, ex vivo cell, etc.
  • a cell may be selected for stable expression of the DBP.
  • a cell may be selected for expressing a threshold level of the DBP.
  • a cell selected for expression of the DBP may be subjected to expansion, freeze/thaw or otherwise prepared for introduction into a subject in need thereof.
  • a cell expressing a DBP either constitutively or in an inducible manner may be administered to the subject, for instance in the circulatory system by means of intravenous delivery or delivery into a solid tissue such as bone marrow.
  • Exemplary mammalian cells can include, but are not limited to, 293A cell line, 293FT cell line, 293F cells , 293 H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CHO- K1 cells, Expi293FTM cells, Flp-InTM T-RExTM 293 cell line, Flp-InTM-293 cell line, Flp-InTM- 3T3 cell line, Flp-InTM-BHK cell line, Flp-InTM-CHO cell line, Flp-InTM-CV-l cell line, Flp- InTM-Jurkat cell line, FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTM 293 MSR cell line, GS-CHO cell line, HepaRGTM cells, T-RExTM Jurkat cell line, Per.C6 cells, T-RExTM- 293 cell line, T-RExTM-CHO cell line, T-RExTM-HeLa cell line, NC
  • a target cell is a cancerous cell.
  • Cancer can be a solid tumor or a hematologic malignancy.
  • the solid tumor can include a sarcoma or a carcinoma.
  • Exemplary sarcoma target cell can include, but are not limited to, cell obtained from alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoi
  • Exemplary carcinoma target cell can include, but are not limited to, cell obtained from anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.
  • CUP Unknown Primary
  • the cancerous cell can comprise cells obtained from a hematologic malignancy.
  • Hematologic malignancy can comprise a leukemia, a lymphoma, a myeloma, a non- Hodgkin’s lymphoma, or a Hodgkin’s lymphoma.
  • the hematologic malignancy can be a T-cell based hematologic malignancy.
  • the hematologic malignancy can be a B-cell based hematologic malignancy.
  • Exemplary B-cell based hematologic malignancy can include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (LL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, s
  • Exemplary T-cell based hematologic malignancy can include, but are not limited to, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), blastic NK- cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related T-cell lymphomas.
  • PTCL-NOS peripheral T-cell lymphoma not otherwise specified
  • anaplastic large cell lymphoma angioimmunoblastic lymphoma
  • ATLL adult T-cell leukemia/lymphoma
  • blastic NK- cell lymphoma enteropathy-type T-cell lymphoma
  • a cell can be a tumor cell line.
  • Exemplary tumor cell line can include, but are not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH/2A, SNU-398, PLHC-1, HepG2/SF
  • Genetic modification can involve introducing a functional gene for therapeutic purposes, knocking out a gene for therapeutic gene, or engineering a cell ex vivo (e.g., HSCs or CAR T cells) to be administered back into a subject in need thereof.
  • a cell ex vivo e.g., HSCs or CAR T cells
  • Cells such as hematopoietic stem cells (HSCs) and T cells, can be engineered ex vivo to express the DBP.
  • nucleic acid encoding the DBP can be directly administered to a subject in need thereof.
  • the target gene may be an endogenous gene such as human fetal gamma globin gene, PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the ECL11A gene, a CELE gene, a TGFER1 gene, a SERPINA1 gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • endogenous gene such as human fetal gamma globin gene, PDCD 1 gene, a CTLA4 gene,
  • compositions disclosed herein may comprise one or more DBP, polynucleotides encoding the DBPs, vectors comprising same, and cell comprising the DBP or polynucleotides encoding the DBPs, as contemplated herein. These compositions are useful for increasing a human g-globin gene in a cell or a population of cells.
  • the cell may be a hematopoietic cell, e.g., a hematopoietic stem or progenitor cell, or a CD34 + cell.
  • the polypeptides described herein may be present in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • the polypeptides are present in a therapeutically effective amount in the pharmaceutical composition.
  • a therapeutically effective amount can be determined based on an observed effectiveness of the composition.
  • a therapeutically effective amount can be determined using assays that measure the desired effect in a cell.
  • compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic methods and uses described herein.
  • compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein.
  • Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, nuclease inhibitors, protease inhibitors, a suitable vehicle such as physiological saline solution or citrate buffered saline.
  • a pharmaceutical composition comprising the DBP as disclosed herein and a pharmaceutically acceptable excipient is provided.
  • a pharmaceutical composition comprising the nucleic acid as disclosed herein or the vector as disclosed herein and a pharmaceutically acceptable excipient is provided.
  • a pharmaceutical composition comprising the host cell of comprising the DBP disclosed herein or a nucleic acid as disclosed herein or the vector as disclosed herein is provided.
  • compositions of the present disclosure find use in a variety of therapeutic and research applications.
  • compositions disclosed herein such as a compsotion comprising a nucleic acid encoding a DBP that displaces the TF ZBTB7A from the fetal g- globin gene promoter and results in expression of fetal hemoglobin- g (HBG) from the fetal g- globin gene, the DBP encoded by the nucleic acid, or a cell comprising the nucleic acid or the DBP may be in a method for the increasing expression of fetal hemoglobin-g (HBG) in a subject in need thereof.
  • the subject may have a blood cell disorder such as a hemoglobinopathy.
  • the subject has sickle cell anemia or thalassemia.
  • compositions comprising the disclosed polypeptides can be delivered into a target cell by any suitable means, including, for example, by contacting the cell with the polypeptide or a nucleic acid encoding the polypeptide.
  • the DBP or a can be delivered into cells in a particular tissue (e.g., a solid tumor) by injecting a composition comprising the positively charged polypeptide directly into the solid tumor.
  • a particular tissue e.g., a solid tumor
  • administration involves systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion), direct injection (e.g., intrathecal), or topical application, etc.
  • systemic administration e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion
  • direct injection e.g., intrathecal
  • topical application e.g., topical application, etc.
  • Nucleic acids encoding the DBPs may be delivered into a target cell by e.g., vectors (e.g., viral or non- viral vectors), non- vector based methods (e.g., using naked DNA or
  • DNA complexes e.g., cationic liposomes, polymeric nanoparticles, polymers, etc.
  • Nucleic acids encoding the DBPs can be delivered directly to cells as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., erythrocytes, HSCs).
  • the nucleic acid vector can also include any suitable number of regulatory/control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES).
  • Viral vectors used for delivering the nucleic acid encoding a DBP include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associate viral vectors, and the like.
  • Nucleic acid encoding DBP or cells comprising such a nucleic acid or cells expressing the DBP can be administered to subjects by any suitable mode or route, whether local or systemic.
  • Systemic modes of administration include oral and parenteral routes.
  • Parenteral routes include, by way of example, intravenous, intramarrow, intrarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes.
  • Nucleic acids administered systemically can be modified or formulated to target, e.g., HSCs, hematopoietic stem/progenitor cells, or erythroid progenitors or precursor cells.
  • Local modes of administration include, by way of example, intramarrow injection into the trabecular bone or intrafemoral injection into the marrow space, and infusion into the portal vein.
  • Administration can be provided as a periodic bolus (for example, intravenously) or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag or implantable pump).
  • Nucleic acids and/or cells can be administered locally, for example, by continuous release from a sustained release drug delivery device.
  • a subject in need of modulation of gene expression by administering a composition as disclosed herein such as in need of increased expression of HBG may have a deficiency of globin synthesis such as thalassemia syndromes and/or structural abnormalities of globin such as sickle cell syndromes and syndromes associated with unstable hemoglobins. These diseases are referred to as hemoglobinopathy.
  • Thalassemia syndromes result from deficiencies in either alpha-globin (a-thalassemia) or beta-like globin (b-thalassemia) chains.
  • a- Thalassemia is symptomatic during gestation, as a-globin is required for fetal hemoglobin (HbF, a2g2).
  • HbF fetal hemoglobin
  • b-thalassemia is asymptomatic until around 6 months after birth. Mutations that cause prolonged production of fetal g-globin chains may present later, at 2 to 4 years of age.
  • the major pathologic process in thalassemia is the imbalance of alpha and non alpha globin chain accumulation.
  • the precipitated a- globin chains are particularly toxic, damaging cell membranes and causing apoptosis.
  • bO-Thalassemias are characterized by a complete absence of any beta globin chains.
  • b-i-Thalassemias are characterized by detectable presence of a reduced amount of beta chains.
  • beta thalassemia major thalassemia intermedia
  • thalassemia minor There are three principal categories of beta thalassemias: thalassemia minor.
  • a person carries the beta thalassemia trait and the hemoglobin S trait (the abnormal hemoglobin found in people with sickle cell disease), which leads to HbS beta thalassemia.
  • the severity of this condition varies according to the amount of normal beta globin produced by the beta globin gene. When no beta globin is produced by the beta globin gene, the condition is almost identical to sickle cell disease.
  • HbS hemoglobin S
  • Sickle cell anemia b8/b8 a common form of sickle cell disease (SCD)
  • HbS Hemoglobin S
  • Additional mutations in the b-globin gene can also cause other abnormalities in b-globin, leading to other types of sickle cell disease.
  • These abnormal forms of b- globin are often designated by letters of the alphabet or sometimes by a name.
  • one b-globin subunit is replaced with HbS and the other b-globin subunit is replaced with a different abnormal variant, such as hemoglobin C (HbC; b-globin allele noted as b ( ) or hemoglobin E (HbE; b-globin allele noted as b E ).
  • HbC hemoglobin C
  • HbE hemoglobin E
  • HbSC hemoglobin SC
  • HbC results from a mutation in the b-globin gene and is the predominant hemoglobin found in people with HbC disease (a.2b ( 2).
  • HbC disease is relatively benign, producing a mild hemolytic anemia and splenomegaly.
  • the severity of HbSC disease is variable, but it can be as severe as sickle cell anemia.
  • HbE is caused when the amino acid glutamic acid is replaced with the amino acid lysine at position 26 in b-globin, noted as Glu26Lys or E26K.
  • People with HbE disease have a mild hemolytic anemia and mild splenomegaly.
  • the HbE mutation is present with HbS.
  • a person may have more severe signs and symptoms associated with sickle cell anemia, such as episodes of pain, anemia, and abnormal spleen function.
  • the subject is a subject who has been diagnosed as having a hemoglobinopathy selected from the group consisting of hemoglobin C disease, hemoglobin E disease, sickle cell anemia, sickle cell disease (SCD), thalassemia, b-thalassemia, thalassemia major, thalassemia intermedia, hemoglobin Bart syndrome and hemoglobin H disease.
  • a hemoglobinopathy selected from the group consisting of hemoglobin C disease, hemoglobin E disease, sickle cell anemia, sickle cell disease (SCD), thalassemia, b-thalassemia, thalassemia major, thalassemia intermedia, hemoglobin Bart syndrome and hemoglobin H disease.
  • the cells may be administered as part of a bone marrow or cord blood transplant in an individual that has or has not undergone bone marrow ablative therapy.
  • cells contemplated herein are administered in a bone marrow transplant to an individual that has undergone chemoablative or radioablative bone marrow therapy.
  • a dose of cells expressing the DBPs is delivered to a subject intravenously.
  • the cells are hematopoietic stem cells which are intravenously administered to a subject.
  • the effective amount of genome edited cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of genome edited cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • HUDEP-2 cells (PMID 23533656) were grown in StemSpan SFEM (Stemcell Technologies) supplemented with 2% PennStrep, 1% L-Glutamine, 1 ug/mL doxy cy cline, 100 ng/mL recombinant human SCF (Peprotech), 3 IU/mL recombinant human EPO (Peprotech), and 10-6 M dexamethasone.
  • TALE transfection mRNA was in vitro transcribed using T7 mScriptTM Standard mRNA Production System (Cellscript) following manufacturer’s instructions and RNA size and concentrations were determined using the Advanced Analytical Fragment Analyzer (Agilent). 5 x 10 5 HUDEP-2 cells were electroporated with 4 ug TALE mRNA and 100 ul BTXpress solution (BTX) in 96-well cuvette (BTX) using the ECM 830 Square Wave Electroporation System (Harvard Apparatus) and HT200 Plate Handler (BTX) with 250mS interval, 250V for 5msec pulse. Electroporated cells were transferred to 12 or 6-well plates, and RNA was harvested 12, 24, or 48 hours later. After the initial time course experiment, all samples were harvested at 48 hours.
  • Donor plasmid contains a splice acceptor site followed by T2A and GFP to utilize an endogenous promoter to drive expression of GFP after integration and an EF1 alpha-driven TALE followed by WPRE (sequences provided below).
  • Cells were maintained in a T-25 flask and media was changed every 24 hours post-transfection for two days. Approximately 7-12 days post-transfection, single cells were sorted for GFP expression into 96-well plates using the MoFlo Astrios (Beckman Coulter). Cells were maintained in 96-well plates for ⁇ 12 days with doxycycline supplemented every two days and media changes on days 8 and 10. Wells containing cells were identified, consolidated, and further maintained.
  • qPCRs were ran with multiplexed Taqman primer/probe sets specific to HBG (Hs00361131_gl, VIC) and HBB (Hs00747223_gl, FAM) from ThermoFisher Scientific, cDNA diluted at a 1:10 ratio, and SsoAdvanced Universal Probes Supermix (Bio-Rad) on a Bio-Rad CFX96 Real Time System. Primer/probe sets were confirmed to be accurate and robust in multiplexing assays as no difference was observed when tested individually or multiplexed.
  • HBG measurements were normalized to measurements of HBB and compared to transfected control cells in biological triplicate. For analysis of stably-integrated TALE HUDEP-2 cell lines, amount of HBG expression was normalized to total HBB and HBG expression, with 2 - 5 measurements taken at different timepoints in culture per sample.
  • EXAMPLE 1 TALES FOR SPECIFIC REGULATION OF A TARGET GENE
  • DNA binding proteins that not only displace an endogenous TL but are specific for a target gene.
  • the DNA binding protein replaces endogenous TL from only a target gene since it binds to a sequence present only in the target gene.
  • TALE proteins that specifically bind to a sequence in a target gene were designed and tested.
  • /-hemoglobinopathies such as, sickle cell disease, //-thalassemia, are caused by mutations in the adult /Z-globin gene.
  • globin genes undergo switching: adult //-globin is expressed and the fetal y-globin promoter is directly bound and silenced by the transcription factors ZBTB7A and BCL11A (Bauer, D. et ak, Blood 2012). Mutations in the fetal y-globin promoter result in incomplete silencing of the fetal y-globin promoter (Hereditary
  • HPPH Persistence of Petal Hemoglobin; HPPH.
  • HPPH patients who inherit sickle cell gene (Hb S) show symptomatic amelioration of sickle cell disease. Accordingly, reactivation of fetal globin expression to increase level of functional Hb is avidly being sought as a therapeutic avenue.
  • TALE proteins that bind to TP binding sites were designed to test the hypothesis that displacing TPs bound to g-globin proximal promoter may reactivate fetal y-globin expression.
  • Pour TALEs (All, Bl, B5, and B7) that bind to different nucleotide sequences spanning the binding site for the TP ZBTB7A and three TALEs (D2, Dll, and El) that bind to different nucleotide sequences spanning the binding site for the TP BCL11A were designed.
  • mRNA encoding the TPs were transfected into HUDEP- 2 cells and relative expression of fetal hemoglobin (HBG) as compared to adult hemoglobin (HBB) at various time points after transfection was measured.
  • HBG fetal hemoglobin
  • HBB adult hemoglobin
  • FIGS. 1A-1B TALEs that replaced the TF BCL11A has no significant effect on HBG expression.
  • HUDEP-2 cell lines expressing All TALE resulted in globin switching at the RNA and protein levels.
  • FIGS. 2A-2D HUDEP-2 cell lines expressing All TALE resulted in globin switching at the RNA and protein levels.
  • FIG. 3 Cassette for expression of TALE designed for integration into the AAVS1 safe harbor locus.
  • FIG. 4 Increased expression of HBG is accompanied by a decreased expression of HBB. This observation is surprising as it has not been previously reported that switching on the expression of HBG has an effect on the expression of HBB.
  • the decreased expression of HBB may be beneficial as only low levels of HBG would be needed to provide functional Hb as compared to other therapeutic strategies that intend to provide high levels of HBG to compete with the HBB expressed a patient.
  • a method for modulating expression of a gene in a cell comprising introducing into the cell a DNA binding polypeptide (DBP) that binds a sequence in regulatory region of a gene bound by a transcription factor (TF), thereby displacing the TF and modulating expression of the gene.
  • DBP DNA binding polypeptide
  • sequence is a sequence that has previously been identified as being associated with activity of the TF based on reduced activity of the TF when the sequence includes a single nucleotide polymorphism (SNP) or a mutation.
  • SNP single nucleotide polymorphism
  • nucleic acid is a deoxyribonucleic acid (DNA).
  • nucleic acid is a ribonucleic acid (RNA).
  • a recombinant DNA binding polypeptide comprising a plurality of repeat units (RUs) ordered from N-terminus to C-terminus of the DBP to bind to a nucleic acid sequence in the fetal g-globin gene promoter, wherein the nucleic acid sequence comprises a sequence bound by the transcription factor (TF) ZBTB7A, wherein each of the RU comprises the sequence Xi-i 1X12X13X14-33, 34, or 35 (SEQ ID NO: 4), wherein:
  • Xi-11 is a chain of 11 contiguous amino acids
  • X14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids
  • X12X13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G);
  • NV or HN for recognition of A or G
  • H* H* , HA, KA, N* , NA, NC, NS, RA, or S* for recognition of A or T or G or C
  • (* ) means that the amino acid at X 13 is absent, wherein the DBP displaces the TF from the fetal g-globin gene promoter and results in expression of fetal hemoglobin- g (HBG) from the fetal g-globin gene.
  • Xi-11 is a chain of 11 contiguous amino acids
  • X 14-20 or 21 or 22 is a chain of 7, 8 or 9 contiguous amino acids
  • X 12 X 13 is selected from:
  • nucleic acid sequence comprises RUs that bind to the nucleotide sequence: CCTCTTGGGGGCCCCTTCCC (SEQ ID NO: 3).
  • nucleic acid of clause 29, wherein the nucleic acid is operably linked to a promoter sequence that confers expression of the DBP.
  • nucleic acid of any one of clauses 29-31 wherein the nucleic acid is a deoxyribonucleic acid (DNA).
  • nucleic acid is a ribonucleic acid (RNA).
  • a host cell comprising the nucleic acid of any of clauses 29-33 or the vector of clause 34 or 35.
  • a pharmaceutical composition comprising the DBP of any of clauses 17-28 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising the nucleic acid of any of clauses 29-33 or the vector of clause 34 or 35 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising the host cell of any one of clauses 36-45.
  • a method for increasing expression of fetal hemoglobin-g (HBG) in a subject in need thereof comprising administering to the subject the pharmaceutical composition of any one of clauses 46-48.

Abstract

La présente invention concerne des procédés et des compositions permettant de moduler l'expression d'un gène cible dans une cellule par réduction de la liaison d'un facteur de transcription endogène à une séquence régulatrice du gène cible. Le procédé comprend l'introduction dans la cellule d'un polypeptide de liaison à l'ADN (DBF) qui lie une séquence dans la région régulatrice d'un gène cible lié par un facteur de transcription (TF), déplaçant ainsi le TF et modulant l'expression du gène cible. Le DBF peut être conçu pour se lier à une séquence comprenant le site de liaison pour le TF et des nucléotides supplémentaires présents sur un ou les deux côtés de la séquence. En conséquence, le DBF se lie spécifiquement à un site de liaison pour le TF dans le gène cible, mais pas dans d'autres gènes qui sont également régulés par la liaison du TF mais ne comprennent pas les nucléotides présents sur un ou les deux côtés de la séquence.
PCT/US2020/055534 2019-10-15 2020-10-14 Protéines de liaison à l'adn permettant de déplacer des facteurs de transcription endogènes liés à des régions régulatrices de gènes WO2021076592A1 (fr)

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US20060182736A1 (en) * 2003-06-10 2006-08-17 Kim Jin-Soo Transducible dna-binding proteins

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WO2013016434A1 (fr) * 2011-07-27 2013-01-31 The Board Institute, Inc. Compositions et méthodes de traitement du cancer de la tête et du cou
US8450107B1 (en) * 2011-11-30 2013-05-28 The Broad Institute Inc. Nucleotide-specific recognition sequences for designer TAL effectors

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US20060182736A1 (en) * 2003-06-10 2006-08-17 Kim Jin-Soo Transducible dna-binding proteins

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FENG WALDO C, SOUTHWOODL CHERIE M, BIEKERLN JAMES J: "Analyses of beta-Thalassemia Mutant DNA Interactions with Erythroid Kriippel-like Factor (EKLF), an Erythroid Cell -specific Transcription Factor", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 2, 1 January 1994 (1994-01-01), pages 1493 - 1500, XP055922351, DOI: 10.1016/S0021-9258(17)42283-6 *
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